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Carboxylesterases, importance for detoxification of organophosphorus anticholinesterases and trichothecenes

Several different types of experiments, including the use of inhibitors, have shown that carboxylesterases are a major factor in the metabolism and therefore detoxification of organophosphorus compounds such as soman and trichothecene toxins. The development of a new assay method for the enzyme has allowed us to separate the carboxylesterases into two major groups. The carboxylesterases can, however, be further separated by gel filtration, affinity chromatography, isoelectric focusing, and chromatofocusing into several isoenzymes. Liver microsomal carboxylesterases can be separated into five or six isoenzymes whereas guinea-pig plasma contains two isoenzymes. The isoenzymes differ in molecular weights, isoelectric points, substrate specificities, and affinity for inhibitors. Intravenous administration of a carboxylesterase preparation lowered the toxicity of soman in young rats. Carboxylesterases from rat and guinea-pig plasma inhibited by soman could be reactivated by DAM, whereas enzymes from porcine liver were not reactivated. Only one of the isoenzymes from rat liver microsomal preparation was responsible for the metabolism of T-2 toxin to HT-2. The further metabolism of HT-2 was performed by esterases from rat liver cytoplasma. Long-term exposure of the bronchial muscle to low concentration of soman modulate the bronchial contraction.

Pharmacological Agents, Hippocampal EEG, and Anticonvulsant Effects on Soman-Induced Seizures in Rats

Changes in the hippocampal theta rhythm were used as a model in which anticonvulsant drugs may be screened for their potential to antagonize soman-induced (1xLD(50)) seizures. The zinc chelator, ethylenediaminetetra acetic acid (EDTA) (300mg/kg), and the NMDA receptor antagonist, HA-966 (60mg/kg), both disrupted the theta rhythm, but did not antagonize soman-induced seizures, neither separately, nor in combination. The anticholinergic and antiglutamatergic procyclidine (6mg/kg) did not influence the theta activity. The GABAergic agonists, diazepam (10mg/kg) and pentobarbital (30mg/kg), both reduced the theta frequency. Procyclidine, diazepam, and pentobarbital did not stop soman-induced seizures when administered separately, but both convulsions and seizure activity terminated when these agents were given together, and the rats slept through the critical convulsion period. This triple therapy was 100% effective, when administered 30-40min following onset of convulsions, and the rats displayed apparently normal behavior the next day. A screening model of potential anticonvulsants cannot be based on alterations in hippocampal EEG activity. Procyclidine, diazepam, and pentobarbital in combination disrupted the theta rhythm like the combination of EDTA and HA-966, but the latter combination did not have anticonvulsant effect. It is concluded that a triple regimen consisting of procyclidine, diazepam, and pentobarbital can effectively terminate soman-induced seizures that have lasted 30min or more.

Acute and sub-acute inhalation of an organophosphate induce alteration of cholinergic muscarinic receptors

Acute and sub-acute inhalation exposure of rats to the organophosphorus compound soman (O-[1,2,2-trimethylpropyl]-methylphosphonofluoridate) reduced the contraction of the bronchial smooth muscle induced by cholinergic stimulation. Acute exposure to 8.51 mg/m3 of soman for 45 min (total dose of 383 mg X min/m3) inhibited the acetylcholinesterase (AChE) activity of the bronchial smooth muscle by 85% and reduced the contraction induced by ACh and carbachol by 70% and 80% respectively. In spite of the extensive inhibition of AChE and reduction in the contraction following cholinergic stimulation, there was no alteration of the binding capacity (Bmax) or the equilibrium dissociation constant (Kd) to [3H]-quinuclidinyl benzilate ([3H]-QNB) in the rat bronchi following such an acute exposure. After sub-acute exposure (40 hr) to 0.45-0.63 mg/m3 of soman (total dose of 1080-1519 mg X min/m3) there was a reduction in AChE-activity of 94% and in the contraction of the bronchial smooth muscle induced by ACh and carbachol of 70%. Furthermore, also a reduction of the binding capacity to [3H]-QNB of approximately 40% was observed. Following exposure to soman by both acute and sub-acute inhalation exposure there was an increase in the apparent affinity (pD2) to ACh in the bronchial smooth muscle, due to the extensive inhibition of the AChE-activity. Inhalation of soman also induced a substantial inhibition of the AChE-activity in the lung (86%), but somewhat smaller inhibition in the hippocampus (70%) and almost no inhibition in the neostriatum (19%). Moreover, it was only in the lung where sub-acute exposure to soman produced a reduction of the binding capacity to [3H]-QNB and the reduction was approximately 50%. The results therefore show that after sub-acute inhalation of a relatively low concentration of the AChE-inhibitor soman, alterations in the number of cholinergic receptors are only observed in the peripheral cholinergic nervous system.

A method for generating toxic vapors of soman: Toxicity of soman by inhalation in rats

A method for administration of highly toxic chemicals by inhalation was developed. The model has three features of special interest: (1) a diffusion cell for producing a constant gas concentration, if necessary for several hours and days, (2) a small rapidly equilibrated inhalation chamber (1100 ml), and (3) complete isolation of the toxic chemicals from the atmosphere. The LCt50 of the anticholinesterase soman [o-(1,2,2 trimethylpropyl)-methyl-phosphonofluoridate] was 400 mg min/m3, registered 24 hr after the end of exposure. The lethal concentration X time of soman was 520 +/- 60 mg min/m3 when exposing the animals until death in the inhalation chamber. The exposure was less than 30 min and the concentration of soman was 21 mg/m3. The inhibition of acetylcholinesterase, cholinesterase, and carboxylesterase activities in different tissues was analyzed to study the possible barrier mechanisms that might exist in the body to soman. There was a large inhibition of the carboxylesterase and cholinesterase activities in bronchi and lungs as well as in blood. Carboxylesterases were important as detoxifying enzymes, as shown by 70% enhancement in toxicity of soman following sc pretreatment with TOCP (tri-ortho-cresyl-phosphate), a carboxylesterase inhibitor.

Enhanced efficacy of anticonvulsants when combined with levetiracetam in soman-exposed rats

Results from studies based on microinfusions into seizure controlling brain sites (area tempestas, medial septum, perirhinal cortex, posterior piriform cortex) have shown that procyclidine, muscimol, caramiphen, and NBQX, but not ketamine, exert anticonvulsant effects against soman-induced seizures. The purpose of the present study was to examine whether levetiracetam (Keppra(®)) may enhance the anticonvulsant potency of the above drugs to become optimally effective when used systemically. Levetiracetam has a unique profile in preclinical models of epilepsy and has been shown to increase the potency of other antiepileptic drugs. The rats were pretreated with pyridostigmine (0.1mg/kg) to enhance survival and received anticonvulsants 20 min after onset of seizures evoked by soman (1.15 × LD(50)). The results showed that no single drug was able to terminate seizure activity. However, when levetiracetam (LEV; 50mg/kg) was combined with either procyclidine (PCD; 10mg/kg) or caramiphen (CMP; 10mg/kg) complete cessation of seizures was achieved, but the nicotinic antagonist mecamylamine was needed to induce full motor rest in some rats. In a subsequent experiment, rats were pretreated with HI-6 (125 mg/kg) to enhance survival and treatment started 40 min following seizure onset of a soman dose of 1.6 × LD(50). LEV (50mg/kg) combined with either PCD (20mg/kg) or CMP (20mg/kg) terminated seizure activity, but the survival rate was considerably higher for LEV+PCD than LEV+CMP. Both therapies could also save the lives of rats that were about to die 5-10 min after seizure onset. Thus, the combination of LEV and PCD or CMP may make up a model of a future autoinjector being effective regardless of the time of application.

Roles of perirhinal and posterior piriform cortices in control and generation of seizures: A microinfusion study in rats exposed to soman

Identification of critical receptors in seizure controlling brain regions may facilitate the development of more efficacious pharmacological therapies against nerve agent intoxication. In the present study, a number of drugs with anticonvulsant potency were microinfused into the perirhinal cortex (PRC) or posterior piriform cortex (PPC) in rats. The drugs used exert cholinergic antagonism (scopolamine), glutamatergic antagonism (ketamine, NBQX), both cholinergic and glutamatergic antagonism (procyclidine, caramiphen), or GABAergic agonism (muscimol). The results showed that in the PRC anticonvulsant efficacy against soman-induced seizures (subcutaneously administered) was achieved by procyclidine or NBQX, but not by ketamine, scopolamine, caramiphen, or muscimol (Experiment 1). Hence, both muscarinic and glutamatergic NMDA receptors had to be antagonized simultaneously or AMPA receptors alone, suggesting increased glutamatergic activation in the PRC before onset of seizures. In the PPC, anticonvulsant effects were assured by scopolamine or muscimol, but not by procyclidine, caramiphen, NBQX, or ketamine (Experiment 2). Thus, muscarinic and GABA(A) receptors appear to be the critical ones in the PPC. Microinfusion of soman into the PRC or PPC resulted in sustained seizure activity in the majority of the rats of both infusion categories. The rhinal structures encompassed in this study apparently have critical functions as both control and trigger sites for nerve agent-evoked seizures.

The threat of mid-spectrum chemical warfare agents.

There is a spectrum of several threat agents, ranging from nerve agents and mustard agents to natural substances, such as biotoxins and new, synthetic, bioactive molecules produced by the chemical industry, to the classical biological warfare agents. The new, emerging threat agents are biotoxins produced by animals, plants, fungi, and bacteria. Examples of such biotoxins are botulinum toxin, tetanus toxin, and ricin. Several bioactive molecules produced by the pharmaceutical industry can be even more toxic than are the classical chemical warfare agents. Such new agents, like the biotoxins and bioregulators, often are called mid-spectrum agents. The threat to humans from agents developed by modern chemical synthesis and by genetic engineering also must be considered, since such agents may be more toxic or more effective in causing death or incapacitation than classical warfare agents. By developing effective medical protection and treatment against the most likely chemical and mid-spectrum threat agents, the effects of such agents in a war scenario or following a terrorist attack can be reduced.

In vitro effects of soman on bronchial smooth muscle.

The in vitro exposure of rat bronchial smooth muscle to the cholinesterase inhibitor soman (O-[1,2,2-trimethylpropyl]-methyl-phosphonofluoridate) potentiated the rapid and concentration dependent increase in the contraction induced by acetylcholine (ACh). There was a substantial increase in the response to ACh when soman was present in concentrations from 10 nM to 1 microM which correspond to a 65-100% inhibition of acetylcholinesterase (AChE). The apparent affinity (pD2) to ACh increased from 3.7 to 6.7 without any change in intrinsic activity (alpha) in this concentration interval. In contrast, soman did not alter the apparent affinity or intrinsic activity of carbachol, which supports the suggestion that the effect of soman is entirely due to its anticholinesterase activity. Soman by itself induced contraction which begun at 1-10 nM. This may be explained from its anticholinesterase activity and the subsequent increase in the synaptic concentration of spontaneously released ACh. The effect of soman on inhibition of cholinesterase and carboxylesterases have also been examined. The results demonstrate that low concentrations of soman induces contraction of the airway smooth muscle.

Behavioral side effects in rats treated with acetylcholinesterase inhibitors suggested used as prophylactics against nerve agents

Acetylcholinesterase inhibitors in combination with an anticholinergic, particularly anticholinergics with antiglutamatergic properties, can effectively protect against nerve agent-induced seizures and lethality. The objective of the present study was to examine potential behavioral side effects of the anticholinesterases physostigmine (0.1mg/kg), galantamine (3mg/kg), huperzine (0.5mg/kg), and donepezil (2.5mg/kg) alone or each drug in combination with anticholinergic procyclidine (3mg/kg). The results showed that rats injected intraperitoneally with galantamine displayed a mild cognitive deficit in terms of reduced preference for novelty that was similarly found among animals treated with procyclidine combined with either galantamine or donepezil. Locomotor activity and rearing were radically depressed in all groups treated with anticholinesterases as well as in combination with procyclidine. Reductions in activity were most prominent for rats injected with galantamine alone. Equalizing effects of cholinesterase inhibitors and anticholinergics were absent in the present context. Findings from previous studies that both systemic and local (amygdala) application of physostigmine cause increased fear-motivated freezing response in rats, may explain the marked reductions in activity among the present rats. In view of these findings, use of anticholinesterases (crossing the blood-brain barrier) as prophylactics against nerve agents must be carefully examined to avoid severe side effects.

Effect of pyridostigmine pretreatment HI-6 and toxogonin® treatment on rat tracheal smooth muscle response to cholinergic stimulation after organophosphorus inhalation exposure

The ex vivo contraction response of the rat tracheal smooth muscle was examined after 10 min in vivo inhalation of soman and/or pretreatment with pyridostigmine and/or post-exposure treatment with HI-6 ([[[(4-aminocarbonyl) pyridinio]methoxy]methyl]-2[(hydroxyimino) methyl]pyridinium dichloride) or Toxogonin® (1,1′-[oxybis-(methylene)]bis[4-[(hydroxyimino)methyl]-pyridinium] dichloride). In vivo pretreatment with pyridostigmine was achieved by subcutaneous (s. c.) implantation of an osmotic pump that delivered pyridostigmine continuously (0.01 mg/h) in the neck region of the rat 18 h before soman exposure. The ex vivo cholinergic tracheal smooth muscle response increased during the first 60 min after soman exposure in animals pretreated with pyridostigmine. The amplitude of the contraction response in pyridostigmine pretreated animals was about 60% of control, compared to 15% of control without pyridostigmine pretreatment. Pyridostigmine pretreatment also produced significant recovery of the total cholinesterase (ChE) activity in plasma, but not in trachea and lung. Intraperitoneal (i. p.) injection of HI-6 or Toxogonin® (50 mg/kg), immediately after 10 min inhalation exposure to soman, also significantly improved the ex vivo cholinergic contraction response of the trachea (decapitation 15 min after oxime administration). The recovery of the physiological response with Toxogonin® was, however, not stable. HI-6 was superior to Toxogonin® with respect to the initial airway contraction response, and the response increased up to a stable level not significantly different from control. There was no significant reactivation of the ChE activity after treatment with the oximes. Combination of pyridostigmine pretreatment and oxime treatment enhanced the recovery of the tracheal contraction response and the ChE activity in the trachea compared to treatment with oximes alone. Experiments with in vitro exposure to soman followed by washout and addition of oximes were also performed. The results show that both oximes effectively re-establish the tracheal response when administered 10 min, but not 30 min, after soman. The effect of Toxogonin® was, however, contrary to the effect of HI-6, not stable. These results correspond to the in vivo exposure experiments. The results from this study indicate that HI-6 produces a more potent and stable recovery of an ex vivo peripheral cholinergic response than Toxogonin® after 10 min inhalation exposure to soman.